Wagner FR et al. (2020), Structure of SWI/SNF chromatin remodeller RSC b...

XB-ART-56893

Nature 2020 Mar 01;5797799:448-451. doi: 10.1038/s41586-020-2088-0.

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Structure of SWI/SNF chromatin remodeller RSC bound to a nucleosome.

Wagner FR , Dienemann C , Wang H , Stützer A , Tegunov D , Urlaub H , Cramer P .

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Chromatin-remodelling complexes of the SWI/SNF family function in the formation of nucleosome-depleted, transcriptionally active promoter regions (NDRs)1,2. In the yeast Saccharomyces cerevisiae, the essential SWI/SNF complex RSC3 contains 16 subunits, including the ATP-dependent DNA translocase Sth14,5. RSC removes nucleosomes from promoter regions6,7 and positions the specialized +1 and -1 nucleosomes that flank NDRs8,9. Here we present the cryo-electron microscopy structure of RSC in complex with a nucleosome substrate. The structure reveals that RSC forms five protein modules and suggests key features of the remodelling mechanism. The body module serves as a scaffold for the four flexible modules that we call DNA-interacting, ATPase, arm and actin-related protein (ARP) modules. The DNA-interacting module binds extra-nucleosomal DNA and is involved in the recognition of promoter DNA elements8,10,11 that influence RSC functionality12. The ATPase and arm modules sandwich the nucleosome disc with the Snf2 ATP-coupling (SnAC) domain and the finger helix, respectively. The translocase motor of the ATPase module engages with the edge of the nucleosome at superhelical location +2. The mobile ARP module may modulate translocase-nucleosome interactions to regulate RSC activity5. The RSC-nucleosome structure provides a basis for understanding NDR formation and the structure and function of human SWI/SNF complexes that are frequently mutated in cancer13.

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Species referenced: Xenopus laevis
Genes referenced: h2ac21 h2bc21

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	Extended Data Figure 2. Cryo-EM analysis of the RSC-nucleosome complex. Related to Figures 1 – 3.a. Representative cryo-EM micrograph of the RSC-nucleosome complex shows homogeneously distributed individual particles.b-d. 2D class averages of the RSC-nucleosome complex (b), the ATPase-nucleosome subcomplex (c) and the nucleosome subcomplex (d).e. Fourier shell correlation plots reveal the overall resolutions of the cryo-EM reconstructions.f. Cryo-EM processing workflow for the reconstructions of the RSC-nucleosome complex, the ATPase-nucleosome subcomplex, and the nucleosome subcomplex. Particle distribution after 3D classifications is indicated below the corresponding map. The final maps are shown in colours. The masks used for focused classifications and refinements are colour coded corresponding to the final maps they were used for. Views are generally rotated by 180° with respect to Figure 1c, left.g. Local resolution estimation of the combined ATPase-nucleosome map as implemented in RELION42. We note that the resolution of the peripheral area with the ATPase module is overestimated.h-j. Angular distribution plot for all particles contributing to the final reconstructions of the RSC-nucleosome complex (h), the ATPase-nucleosome subcomplex (i) and the nucleosome subcomplex (j).
	Extended Data Figure 3. Cryo-EM analysis of the free RSC complex. Related to Figures 1 – 3.a. Representative cryo-EM micrograph of the free RSC complex shows homogeneously spaced individual particles.b. 2D class averages of the free RSC complex.c. Cryo-EM processing workflow for the reconstruction of the free RSC complex. Particle distribution after 3D classifications is indicated below the corresponding map. The final maps after focused 3D refinement and masks are depicted in colour. Views are generally rotated by 180° with respect to Figure 1c, right.d. Angular distribution plot for all particles contributing to the final reconstruction of the free RSC complex.e. Two views of the combined RSC core map coloured according to the local resolution as implemented in RELION42.f. Fourier shell correlation plots of the maps used for model building of the RSC core complex.
	Extended Data Figure 4. Cryo-EM densities for selected RSC regions. Related to Figures 1 – 3.a-c. Examples of map quality. Close-up of the Rsc4 β-sheet shows clear separation of individual strands (a). The high quality of the map for the ZZ zinc finger of Rsc8 allowed backbone tracing and placement of side chains as well as for the zinc ion (b). Coiled coil helices of the two Rsc8 subunits with density for one helix (c).d. View along the exit DNA in the direction of the nucleosome showing the low pass-filtered maps for the modules ATPase, ARP, DIM, arm, body, and the nucleosome. At the site where the H2A C-terminal tail protrudes from the nucleosome near Sfh1, low resolution density connecting the arm module and the nucleosome is visible. Density bridging form the ARP module to the exit DNA close to the H3 histone tail can be observed.e. Density representing the finger helix (green) at the acidic patch of the nucleosome (indicated by H2A in yellow). Side chain density is visible for conserved arginine residues.f. Interaction of RSC with the nucleosome is sterically impaired by the flexibly bound ubiquitin moiety at H2B lysine 123 (human K120). The Sfh1 finger helix and the ubiquitin moiety (ubiquitylated nucleosome PDB code 6NOG)75 overlap after superposition of nucleosomes.
	Extended Data Figure 5. Structure of RSC body and arm modules, cancer mutations and remodeler families. Related to Figure 1.a. Cartoon representation of RSC core viewed as in Figure 1. Important structural elements are labelled.b. Conservation between SWI/SNF complexes RSC (yeast) and PBAF (human). Residues that are identical (blue) or conserved (light blue) in human PBAF highlighted on the RSC structure (grey). Purple spheres depict identical residues that show missense mutations in various cancers (Methods).c. Comparison of overall structure of RSC with complexes of INO80 (yeast INO8076) and CHD (yeast CHD177) families. ATPase motor domains are shown in orange, DNA in blue. With regard to the INO80 family, the ATPase of the SWR1 complex also binds SHL +278, whereas the ATPase of the INO80 complex binds SHL –676,79. The ARP module of INO80 contacts exit DNA, which is not the case in RSC. The INO80 complex also contacts both faces of the histone octamer76, resembling the sandwiching interactions made by RSC on a topological level. With respect to the CHD family, the ATPase motor of yeast Chd1 also binds SHL +2, but its DNA-binding region engages with exit DNA near the nucleosome, leading to a different DNA trajectory77,80. With respect to the ISWI family, the ATPase motor binds SHL +281, but other interactions have not been structurally resolved (not shown).
	Extended Data Figure 6. Course of polypeptide chains of architectural subunits Sth1, Rsc8, Rsc58 and ATPase-nucleosome interactions. Related to Figure 1.a. Back view of RSC. The Sth1 subunit of RSC starts with its N-terminus in the body module and tracks through it turning around with a contact helix and loop. Forming the central helix I, the hook and the central helix II it folds back and forth tightly interweaving the body module before it exits with its HSA region through the ARP module to build the ATPase module.b. RSC with the domains of the two Rsc8 subunits highlighted in blue. Both Rsc8 start N-terminal with their SWIRM domains in the arm module where they support the two repeat domains of Sfh1 in a similar manner. They then follow distinct paths through the arm towards the body module where they contribute with both their SANT and ZZ zinc finger domains. Here the two domains of each subunit form different contacts with various interactions partners and whereas one ZZ zinc finger domain is tightly packed at the body and DNA-interaction module interface, the other seems to extend from the body, presumably as additional interaction surface. Both Rsc8 subunits unite again with their C-terminal long helices in a coiled coil fold on the opposite side of the body module.c. Rsc58 N-terminal bromodomain attaches to the top of the body module. Then, Rsc58 follows an interwound path through the body module via the central and connector loop. It turns back docking to the body with a 3-helix bundle and stabilizing the module with its C-terminal end.d. Contacts of Sth1 ATPase motor (orange) with the nucleosome. View as in Fig. 1c, left, but rotated by 45° around a horizontal axis. Arrows indicate directionality of DNA translocation.
	Extended Data Figure 7. DNA recognition and NDR formation. Related to Figure 1.a. Space-filling RSC-nucleosome structure with DIM (green) and SnAC (orange) densities. View on the top as in Fig. 1c, left, but rotated by 90° around the vertical and horizontal axis. Arrows indicate directionality of DNA translocation. Number of upstream DNA base pairs relative to SHL –7 is provided.b. Schematic of a promoter before (top) and after (bottom) RSC remodelling shows NDR formation by sliding the flanking –1 and +1 nucleosomes away from the NDR center. Arrows indicate the transcription start site.
	Extended Data Figure 8. Sequence alignments for the Sth1 ATPase domain and HSA region. Related to Figures 1 – 3.a. Sequence alignment of the S. cerevisiae Sth1 ATPase domain to the homologous Snf2 ATPase domain of the same organism. Secondary structure elements are represented in orange according to the cryo-EM structure of the Snf2 ATPase (PDB entry 5Z3U)17. Residues modelled in the Snf2 structure are topped by a back line with helical regions shown as cylinders and sheet regions as arrows. The Sth1 residues modelled in this work are indicated with a black dashed line below. ATPase motifs are underlined. Invariant residues are coloured in dark blue and conserved residues in light blue. The alignment was generated with MSAProbs71 within the MPI Bioinformatics Toolkit60 and visualized using ESPript82.b. Sequence alignment of the HSA regions from S. cerevisiae homologues Sth1 and Snf2. Illustration and generation of the alignment as in (a).
	Figure 1. RSC-nucleosome complex structure.a. Two views of the low pass-filtered cryo-EM density reveal the overall architecture. The five RSC modules are in different colours. The nucleosome with exit DNA is in yellow. DIM, DNA-interaction module.b. RSC subunit domain architecture. Domain boundaries marked with residue numbers. Black bars indicate modelled regions. HSA, helicase-SANT-associated; SnAC, Snf2 ATP coupling; bromo, bromodomain; armadillo, armadillo repeat fold; RFX, DNA-binding RFX-type winged-helix; SWIRM, Swi3 Rsc8 Moira; ZZ, ZZ-type zinc finger; SANT, Swi3 Ada N-Cor TFIIIB; Zn, Zn(2)-C6 fungal-type zinc finger; RPT, repeat; BAH, bromo-adjacent homology.c. Cartoon representation. Unassigned elements in grey. Mobile domains depicted schematically. Arrows indicate directionality of DNA translocation.
	Figure 2. RSC ATPase and ARP modules.View along the nucleosome dyad (black oval). View as in Fig. 1c, right, but rotated by 45° around a horizontal axis.
	Figure 3. RSC sandwiches the nucleosome.a. RSC-nucleosome interactions viewed along the nucleosome dyad (black oval). On the outer face of the histone octamer, densities for the Sth1 SnAC domain and the histone H4 tail are shown as an orange surface and a green mesh, respectively. On the inner face, the arm module and Sfh1 finger helix are depicted.b. Interaction of the Sfh1 finger helix with the acidic patch of the inner face of the histone octamer (surface representation coloured by electrostatic charge; red, negative; blue, positive). Conserved arginine residues are depicted. Residues mutated in cancer (Methods) highlighted in purple.c. Sequence alignment of the finger helix region (green cylinder) in S. cerevisiae’s (Sc) Sfh1 with its homologs H. sapiens (Hs) BAF47, M. musculus (Mm) BAF47, D. melanogaster (Dm) SNR1 and ScSnf5. Invariant and conserved residues highlighted in dark and light blue, respectively. Yellow boxes contain arginine residues shown in (b). Purple dots mark residues mutated in cancer (Methods).

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